CN111333638A - 18F-labeled isoquinolino pyridazinone compound and synthesis method and application thereof - Google Patents

Abstract

The invention discloses ¹⁸ F-labeled isoquinoline pyridazinone compounds, a synthesis method and application thereof, and belongs to the field of imaging agents. The invention splices and optimizes the isoquinoline pyridazinone compounds through the principle of medicinal chemistry. , on the basis of not changing the structure of the active group of the key drug action, a brand-new organic small molecule targeting the mitochondrial membrane of the myocardium is prepared, and the ¹⁸ F radioisotope is labeled through the positron nuclide labeling experiment; ¹⁸ F-labeled isoquinolinopyridazinone compounds have good myocardial targeting properties and are excellent myocardial imaging agents.

Description

18F-labeled isoquinolinopyridazinone compounds and their synthetic methods and applications

technical field

The invention belongs to the field of imaging agents, and in particular relates to ¹⁸ F-labeled isoquinolinopyridazinone compounds and a synthesis method and application thereof.

Background technique

According to the World Health Organization, the American Heart Association and the National Center for Cardiovascular Diseases, cardiovascular diseases (CVDs) are the leading cause of death in the world today. Nuclear cardiology SPECT and PET stress myocardial perfusion imaging have been fully affirmed by relevant guidelines such as the American College of Cardiology/American Heart Association/American Society of Nuclear Cardiology (ACC/AHA/ASNC). It predicts the risk of sudden cardiac death or non-fatal myocardial infarction, reduces the occurrence of malignant cardiac events, and selects appropriate patients for revascularization or drug therapy. The current clinical radionuclide myocardial imaging is mainly based on SPECT imaging based on the ^99m Tc-MIBI myocardial radionuclide probe.

PET myocardial perfusion imaging has the following advantages over SPECT myocardial perfusion imaging: (1) PET has the technical advantages of higher spatial resolution and more accurate attenuation correction, which can accurately detect the presence of small coronary lesions; (2) can be quantitatively The amount of imaging agent uptake by the myocardium was determined. Therefore, PET myocardial perfusion imaging is more promising than SPECT myocardial perfusion imaging.

Three PET myocardial perfusion imaging agents currently available in clinic, including [ ¹³ N]NH3 (half-life: 9.97min), ⁸² Rb (half-life: 1.27min) and [ ¹⁵ O]H2O (half-life: 2.04min), have too short half-lives, The need for an online cyclotron and the inability to perform exercise stress-gated myocardial perfusion imaging limit the widespread clinical application of PET myocardial perfusion imaging. The development of new myocardial perfusion imaging agents has received extensive attention from researchers at home and abroad. Among them, the physical half-life of ¹⁸ F is as long as 109.8min, which is more suitable for clinical imaging and is the first choice for developing new PET positron drugs. The research on ¹⁸ F-labeled myocardial perfusion imaging agent is the most in-depth. Currently widely reported ¹⁸ F-labeled PET myocardial perfusion imaging agents are mainly divided into two categories: (1) ¹⁸ F-labeled pyridazinone compounds; (2) ¹⁸ F-labeled lipophilic cationic compounds such as quaternary ammonium salts and Quaternary phosphonium salt compounds. Although the former has higher initial myocardial uptake, it has poor in vitro and in vivo stability; while the latter not only has poor in vitro and in vivo stability, but also has high bone uptake due to severe defluorination imaging, which requires further research. Therefore, up to now, the ¹⁸ F-labeled myocardial perfusion imaging agents are all in the preclinical research stage, and no drug has been approved by the US FDA or China's NMDA for clinical application. In conclusion, an ¹⁸ F-labeled isoquinolinopyridazinone compound and its synthesis method and application need to be studied.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention aims to provide ¹⁸ F-labeled isoquinolinopyridazinone compounds, a synthesis method and application thereof, and the ¹⁸ F-labeled isoquinolinopyridazinone compounds of the present invention have better myocardial targeting.

In order to achieve the above-mentioned purpose, the technical scheme adopted in the present invention is:

¹⁸ F-labeled isoquinolinopyridazinone compounds, its structural formula is:

Wherein, n=1-4, X is an O or C atom, R ₁ is a hydrogen atom or an alkyl group, and R ₂ is a halogen atom.

Further, the alkyl group in R ₁ is any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl.

Further, R ₂ is any one of F, Cl and Br.

Further, the synthetic method of ¹⁸ F-labeled isoquinoline pyridazinone compounds comprises the following steps:

(1) take the compound of formula I and LG-(CH ₂ -X)n-LG under basic conditions to generate the precursor compound of formula II;

Wherein, LG is a leaving group, n in LG-(CH ₂ -X)n-LG is an integer greater than 0, and X is an O or C atom;

(2) the precursor compound of formula II reacts with ¹⁸ F ion nucleophilic substitution to generate ¹⁸ F-labeled isoquinolinopyridazinone compounds;

Further, using the positron nuclide labeling method of nucleophilic substitution reaction, that is, using the phase transfer reagent K2.2.2 as the catalyst and acetonitrile as the solvent, the compound of formula II can be heated and reacted for 10-20 minutes to obtain ¹⁸ F. labelled isoquinolinopyridazinones, followed by purification of the product by HPLC.

Further, in step (1), LG is -OTs or -OTf.

Further, the application of ¹⁸ F-labeled isoquinolinopyridazinone compounds in myocardial imaging agents.

Preferably, the method for synthesizing ¹⁸ F-labeled isoquinolinopyridazinone compounds comprises the following steps:

Using compound III as a raw material, it was docked with p-toluenesulfonyl-protected ethylene glycol (ethane-1,2-diyl bis (4-methylbenzenesulfonate, compound IV) in alkaline conditions to generate a precursor for positron nuclide labeling. Compound V.

Precursor compound V ^{reacts with18F} ions to generate18F-labeled positron nuclide probe (compound VI).

The present invention analyzes the structural properties of isoquinoline pyridazinone compounds and positron nuclide myocardial imaging agent 18F-flurpiridaz, and splices and optimizes them through the principle of medicinal chemistry, without changing the activity of key drugs. Based on the structure of the group, a new organic small molecule targeting the mitochondrial membrane of the myocardium is prepared. The small molecule enters the mitochondria through two mechanisms and achieves selective concentration: 1. The positively charged center can pass the potential of the inner and outer mitochondrial membranes. Poor entry into mitochondria (confirmed by experiments); 2. It possesses the parent nucleus structure of flurpiridaz, which can selectively bind to mitochondrial complex I through a mechanism similar to that of flurpiridaz. Through the positron nuclide labeling experiment, we successfully labeled the ¹⁸ F radioisotope. The pharmacological properties of the radiolabel in vivo, such as absorption, distribution, metabolism and excretion, were studied by imaging experiments in normal animals. A rat myocardial ischemia model was established, and the value of this molecule in evaluating myocardial cell survival was studied through animal experiments.

Through research, it is found that ¹⁸ F-labeled isoquinolinopyridazinone compounds have good myocardial targeting, and the mechanism is to selectively accumulate in the mitochondria of myocardial cells through active transport of the potential difference between the inner and outer membranes of myocardial mitochondria. The positron nuclide myocardial imaging agent ¹⁸ F-flurpiridaz is a mitochondrial complex I inhibitor that selectively condenses cardiomyocytes by specifically binding to mitochondrial complex I, which is abundant in cardiomyocytes.

Beneficial effects of the present invention:

The ¹⁸ F-labeled isoquinoline pyridazinone compounds of the present invention have good myocardial targeting properties and can be used as myocardial imaging agents, and the mechanism is to achieve selective concentration in the myocardium through active transport of the potential difference between the inner and outer membranes of myocardial mitochondria. Cell mitochondria; at present, there is no myocardial perfusion imaging agent labeled with ¹⁸ F positron nuclide for clinical use, and most of the myocardial perfusion imaging agents in the research stage are not stable and are easy to decompose when entering the body. The isoquinoline involved in the present invention Pyridazinone myocardial imaging agents not only have a high 'heart-liver' ratio, which can better reflect the function of the heart, but also have high stability, and can provide more accurate myocardial imaging in clinical applications. Functional data for cardiac function assessment.

Description of drawings

Figure 1 is the "time-uptake" curve of compound 12 in different cells;

Figure 2 is the PET imaging of compound 12 in normal animals (Wistar rats);

Figure 3 is a PET imaging of compound 12 in an animal model of myocardial ischemia (Wistar rats).

Detailed ways

In order to further illustrate the technical effect of the present invention, the present invention will be described in detail below through embodiments.

The specific preparation method of ¹⁸ F-labeled isoquinolinopyridazinone compounds is as follows:

Compound 1N-Boc-4-piperidone (10 g, 50 mmol) was dissolved in 80 ml of anhydrous toluene, p-toluenesulfonic acid monohydrate (0.3 g, 1.5 mmol) and tetrahydropyrrole (4.3 g, 60 mmol) were added, and heated Reflux the reaction, use an oil-water separator to separate the water produced by the reaction, cool to room temperature after 3 hours, add p-toluenesulfonic acid monohydrate (0.3 g, 1.5 mmol) and ethyl glyoxylate (50% toluene solution) (11 ml) , 55 mmol), heated to reflux for 2 h, cooled to room temperature, concentrated the reaction solution to about 20 ml, slowly added 4M hydrochloric acid (54 ml) dropwise with vigorous stirring, and continued stirring at room temperature overnight. The organic phase was separated, the aqueous layer was extracted three times with ethyl acetate, the organic layers were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by silica gel column chromatography to obtain compound 2(E)-(1-tert-butyl). Oxycarbonyl-4-oxo-piperidin-3-ylidene)-ethyl acetate 1.5g, 10.6% yield in two steps; and (Z)-(1-tert-butoxycarbonyl-4-oxo-piperidine- 3-ylidene)-ethyl acetate 4.2 g, the two-step yield is 29.7%.

-: 1H NMR(400MHz, CDCl3)δ6.66(t,J=2.5Hz,1H),4.88(d,J=2.5Hz,2H),4.24(q,J=7.2Hz,2H),3.77(t , J=6.3Hz, 2H), 2.65(t, J=6.3Hz, 2H), 1.48(s, 9H), 1.32(t, J=7.1Hz, 3H).

-: 1HNMR(400MHz, CDCl3)δ7.80(s,1H),4.15(q,J=7.1Hz,2H),3.99(t,J=7.4Hz,2H),3.17(d,J=0.8Hz, 2H), 2.74–2.35(m, 2H), 1.54(s, 9H), 1.26(t, J=7.1Hz, 3H).

Compound 2(E)-(1-tert-butoxycarbonyl-4-oxo-piperidin-3-ylidene)-ethyl acetate (1.5 g, 5.3 mmol) was dissolved in 10 ml of absolute ethanol, and tert-butyl Hydrazine hydrochloride (1.3g, 10.6mmol), acetic acid (1.3ml), heated to reflux for 3h, spin-dried the solvent, purified and separated by silica gel column chromatography to obtain 1.1g of pale yellow solid (compound 3), yield 69%.

-: 1HNMR(400MHz, CDCl3)δ12.45(s,1H),6.74(d,J=1.6Hz,1H),4.63(d,J=6.0Hz,2H),3.70(t,J=6.1Hz, 2H), 2.84(t, J＝6.1Hz, 2H), 1.52(s, 9H), 1.49(s, 9H).

Compound 3 (1.0 g, 3.3 mmol) was dissolved in 8 ml of dichloromethane, trifluoroacetic acid (2 ml) was added dropwise at 0°C, the reaction was completed at room temperature for 2 h, and the solvent was spin-dried to obtain 0.6 g of crude compound 4 in yield. 90% was used in the next reaction without further purification. Compound 4 (0.6 g, 2.9 mmol), the product of the previous step, was dissolved in 30 ml of chloroform, 4A molecular sieves (5 g) and C70 (2.4 mg, 2.9 umol) were added, and oxygen was added to the reaction system for 30 seconds. Under irradiation for 3 hours, the reaction solution was cooled to 0 degrees, trimethylsilyl cyanide (0.44 g, 4.4 mmol) was added, and the reaction was raised to room temperature for 4 hours. After monitoring the completion of the reaction by TLC, the reaction was processed and purified and separated by silica gel column chromatography to obtain 0.4 g of compound 5, yield 60%.

-: 1H NMR (400MHz, CDCl3) δ6.33 (d, J=8.8Hz, 1H), 4.31 (s, 1H), 2.47-2.63 (m, 2H), 2.1 (t, J=6.1Hz, 2H) ,1.47(s,9H).

Add compound 6 phthalate (15g, 108mmol) and paraformaldehyde (6.5g, 2155mmol) into a 250ml round bottom flask, cool down to 0 degrees, and then slowly add 33% hydrobromic acid in glacial acetic acid (31ml) It was added dropwise to the reaction system, and after completion of the reaction at room temperature for 20 hours, the temperature was raised to 65° C. and the reaction was performed for 1 hour. The reaction solution cooled to room temperature was poured into ice water, filtered and dried to obtain 22.7 g of a white solid (compound 7) in a yield of 65%.

-: 1HNMR(400MHz, CDCl3)δ6.78(s,2H),4.57(s,4H),3.87(s,6H).

Compound 7 1,2-dibromomethyl-4,5-xylylene ether (600 mg, 1.87 mmol) and DIPEA (0.31 ml, 1.87 mmol) were dissolved in 5 ml of tetrahydrofuran, heated under reflux for 24 h, and then 5 ml of compound A solution of 5 (435 mg, 1.87 mmol) in tetrahydrofuran was added to the reaction system, and the heating was continued for 24 h after completion. After the reaction was completed, the solvent was spin-dried, a 1:1 solution of diethyl ether/methanol was added to the residue, and a solid was precipitated, which was filtered to obtain 410 mg of compound 8 with a yield of 60%.

-: 1H NMR (400MHz, DMSO-d6) δ10.25(s, 1H), 8.23(s, 1H), 7.02(d, J=8.8Hz, 1H), 6.88(d, J=8.8Hz, 1H) , 5.92(s, 1H), 4.15(t, J=6.5Hz, 2H), 3.85(s, 6H), 1.9(t, J=6.5Hz, 2H), 1.47(s, 9H).

Compound 8 (500 mg, 1.37 mmol) was added to a 50 ml round-bottomed flask, heated to 170°C, reacted under reduced pressure with an oil pump for 1.5 h, purified and isolated by silica gel column chromatography to obtain 310 mg of compound 9 with a yield of 65%.

-: 1H NMR (400MHz, DMSO-d6) δ 10.21(s, 1H), 8.20(s, 1H), 7.05(d, J=8.9Hz, 1H), 6.98(d, J=8.9Hz, 1H) , 5.93(s, 1H), 5.35(s, 1H), 4.18(t, J=6.4Hz, 2H), 3.83(s, 3H), 1.9(t, J=6.4Hz, 2H), 1.47(s, 9H).

Compound 9 (300 mg, 0.85 mmol) was dissolved in 10 ml of acetonitrile, potassium carbonate (176 mg, 1.28 mmol) and compound 10 (393 mg, 1.02 mmol) were added, and the reaction was heated at 70 degrees overnight. After the reaction was completed, it was cooled to room temperature and filtered. , the solvent was spin-dried, purified and isolated by silica gel column chromatography to obtain 330 mg of compound 11 with a yield of 68%.

-: 1H NMR (400MHz, DMSO-d6) δ10.27(s, 1H), 8.26(s, 1H), 7.48(d, J=8.1Hz, 2H), 7.41(d, J=8.1Hz, 2H) ,7.05(d,J=9.0Hz,1H),6.98(d,J=9.0Hz,1H),5.90(s,1H),4.42–4.33(m,2H),4.18(t,J=6.4Hz, 2H), 4.06(s, 3H), 3.83-3.75(m, 2H), 3.83(s, 3H), 2.33(s, 3H), 2.06-2.02(m, 2H), 1.9(t, J=6.4Hz ,2H),1.47(s,9H).

Compound 9 (300 mg, 0.85 mmol) was dissolved in 10 ml of acetonitrile, potassium carbonate (176 mg, 1.28 mmol) and compound 13 (237 mg, 1.02 mmol) were added, and the reaction was heated at 70 degrees overnight. After the reaction was completed, it was cooled to room temperature and filtered. , the solvent was spin-dried, purified and separated by silica gel column chromatography to obtain 280 mg of compound 14 with a yield of 80%.

-: 1H NMR (400MHz, DMSO-d6) δ10.25(s, 1H), 8.25(s, 1H), 7.02(d, J=9.0Hz, 1H), 6.88(d, J=9.0Hz, 1H) ,5.93(s,1H),4.20-4.10(m,2H),4.02-4.12(m,4H),3.83(s,3H),1.89-1.95(m,2H),1.52-1.58(m,2H) ,1.48(s,9H).

The ¹⁸ F-HF solution produced by the accelerator was transported to a shielded thermal chamber through a liquid pipeline and captured by a strong cation exchange column, and the 18 F ions adsorbed on the cation exchange column were eluted with a crown ether (K222-K ₂ CO ₃ ) eluent. Elute into a glass reaction tube and heat to remove _H2O . Compound 11 (2 mg) was dissolved in 2 ml of acetonitrile, passed into a reaction tube, and heated at 90 degrees to react for 20 minutes. After the reaction is completed, use 5ml of 30% MeCN and 70 of deionized water mixture containing 0.1% trifluoroacetic acid to dilute, and use HPLC for separation, the adopted chromatography system is: Agilent 1100HPLC system, Waters SunFire Prep C18 5um (250*10mm) column, 30% MeCN-70% _H2O (0.1% TFA), 5 ml/min. A radioactive peak appeared in about 15 minutes, and the radioactive peak was collected and subjected to solvent replacement to wash away the MeCN for subsequent experiments.

Cardiomyocyte uptake experiment: Select primary rat cardiomyocytes, cardiac fibroblasts and H1975 lung cancer cell line respectively, culture the cells to the plateau phase, resuspend and count after collection, and inoculate them in a cell culture plate at a certain concentration overnight for the next One-stage use. The uptake experiment is briefly described as follows: after adding the positron marker to be tested and co-incubating the cells for different time points, the cells are centrifuged, and the radioactivity counts of the cells and the culture medium are measured by a gamma counter, respectively, and the ratio of the uptake of the radioactive marker by the cells is obtained. , plot the "time-radioactive uptake ratio" curve, see Figure 1. The experiment will last for 2 hours.

As shown in Figure 1, compound 12 showed a regular increase in uptake over time in primary rat cardiomyocytes, and gradually reached equilibrium (the curve flattened) after 90 minutes, with a maximum uptake rate of 2.15%. However, the uptake trend of this compound in cardiomyocytes and H1975 lung cancer cells is not as obvious as that in primary cardiomyocytes (which may be related to the abundance of mitochondria in cardiomyocytes), with the highest uptake rates in these two cells being 1.2% and 1.10%.

Normal rat PET imaging experiment: After completing the preparation of positron markers, select healthy normal Wistar rats, anesthetize with isoflurane gas, and perform tail vein injection at a dose of 0.16mCi/Kg (the maximum injection volume does not exceed 1ml), and a static Micro PET/CT scan was performed, and the scan time point was 90 minutes.

The PET imaging images of normal rats are shown in Figure 2. 90 minutes after the drug was injected into the tail vein, the drug was concentrated in the heart, liver and intestine. Among them, the uptake around the heart and the blood pool is low, which can better display the characteristics of the heart. The standard uptake value (SUV) calculated by the imaging software is 3.5. The liver is the main metabolic organ of the drug, so its radioactive concentration is high, and its standard uptake value is 1.2. The intestinal radioactivity is mainly caused by the liver-enteric circulation; the blood uptake value in the left ventricle is used to calculate the blood pool. SUV, which has a standard uptake value of 1.1. It can be seen that the compound can be better concentrated in the myocardium and fully display the heart. At the same time, the ratio of myocardial uptake to liver uptake ('heart-liver ratio') and the ratio of myocardial uptake to blood pool uptake ('heart-to-liver ratio') Heart-to-blood ratio') were 2.91 and 3.18, respectively, which proved that the imaging agent had high selective uptake in the heart, and at the same time, it could achieve better contrast with surrounding organs and tissues, and could be used as a tool to evaluate myocardial vitality.

PET imaging experiment of myocardial ischemia rats: After the preparation of positron markers, Wistar rats with myocardial ischemia were selected, and after anesthesia with isoflurane gas, tail vein injection was performed at a dose of 0.16 mCi/Kg ( The maximum injection volume does not exceed 1ml), and a static Micro PET/CT scan is performed, and the scan time point is also set to 90 minutes.

The PET imaging images of rats with myocardial ischemia are shown in Figure 3. 90 minutes after the drug was injected into the tail vein, the drug was also concentrated in the heart, liver and intestine. Among them, the uptake around the heart and the blood pool is low, which can better display the characteristics of the heart. The SUV calculated by the imaging software is 2.2. The liver is the main metabolic organ of drugs, so its radioactive concentration is high, and the intestinal radioactivity is mainly caused by the liver-enteric circulation. Compared with the SUV (3.5) of normal rat myocardial imaging, the SUV of ischemic myocardium was 2.2, indicating a certain decrease in uptake value. At the same time, the imaging range of the myocardium of the model animals is also small, and the volume of isotope concentration is much smaller than that of the myocardium of normal animals PET imaging. During imaging, we also observed that at 120 minutes, the concentration of radioactivity in the myocardium was much smaller than in normal animals. In conclusion, the imaging parameters of compound 12 in normal animals and myocardial ischemia animals are quite different, which can be used as a powerful tool for evaluating myocardial ischemia.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the technical solutions of the present invention have been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be The scheme is modified or equivalently replaced without departing from the spirit and scope of the present invention, and should be included in the protection scope of the present invention.

Claims (6)

1.¹⁸F-labelled isoquinolinopyridazinonesThe compound is characterized in that the structural formula is as follows:

wherein n is 1-4, X is O or C atom, R₁Is a hydrogen atom or an alkyl group, R₂Is a halogen atom.

2. The compound of claim 1, wherein R is₁The alkyl group in (1) is any of methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl.

3. The compound of claim 1, wherein R is₂Is any one of F, Cl and Br.

4. A method for the synthesis of a compound according to any one of claims 1 to 3, characterized in that it comprises the following steps:

(1) taking a compound of formula I and LG- (CH)₂-X) n-LG in basic conditions to produce a precursor compound of formula II;

wherein LG is a leaving group LG- (CH)₂-X) n-LG wherein n is an integer greater than 0 and X is an O or C atom;

(2) precursor compounds of formula II and¹⁸nucleophilic substitution of F ion to form¹⁸F-labeled isoquinolinopyridazinones;

5. the method of claim 4, wherein in step (1), LG is-OTs or-OTf.

6. The method of claim 1¹⁸The application of the F-labeled isoquinolin pyridazinone compound in a myocardial imaging agent.

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